 Why Radiance?
The Radiance™ Cloning and Expression System provides expression vectors for efficient, directional cloning of PCR products and their subsequent expression in bacterial, insect, and mammalian cells. The system offers an alternative to recombinase- mediated cloning with the unique advantage of removal of all amino-terminal vector-encoded sequences after purification. Radiance Means LIC
for efficient, directional cloning,
expression, and complete fusion tag removal LIC (Ligation-independent Cloning) Vector Kits offer rapid, directional cloning of PCR products without restriction enzyme digestion or ligation reactions (1, 2). The LIC method takes advantage of the 3´→ 5´ exonuclease activity of T4 DNA polymerase to create very specific 12- to 15-nucleotide single-stranded overhangs in the vector and the insert, so that the vast majority of annealed products consists of the desired molecules. The annealed LIC vector and insert are transformed directly into competent E. coli cells, and covalent bonds are formed at the vector-insert junctions within the cell to yield circular plasmid. Directional cloning of the insert is achieved with minimal nonrecombinant background, and cloning is so efficient that virtually all of the resulting colonies contain the desired recombinant. PCR products with complementary overhangs are created by building appropriate 5´-extensions into the primers. The purified PCR products are treated with LIC-qualified T4 DNA polymerase in the presence of the appropriate dNTP to generate the specific vector-compatible overhangs. Ek/LIC Vector Kits
Kits containing Ek/LIC vectors are available for cloning into pET, pRSF, pCDF, pIEx™, pBAC™, or pTriEx™-4 vectors. The LIC site is designed to enable removal of all vector-encoded sequences from the target protein by digestion with Recombinant Enterokinase. The Ek/LIC Vector Kits provide the necessary reagents for creating single-stranded overhangs, annealing with the vector, and transforming competent E. coli cells. Each kit provides enough reagents for 20 annealings and transformations. A Control Insert is included to verify performance. Features - Directional cloning
- No restriction digestion or ligation
- Ultra-low nonrecombinant background; > 95% recombinants
- Same LIC site can be used for insertion into multiple vectors (primers or PCR products)
- Clone directly into 15 expression vectors
- LIC site encodes an enterokinase cleavage signal for complete removal of amino-terminal fusion tag(s) from expressed protein
| Primer design for expression of inserts in Ek/LIC Vectors | Ek/LIC for enterokinase cleavage The Ek/LIC site in Ek/LIC vectors has a 13-base single-stranded overhang upstream of the insert and a 14-base single-stranded overhang downstream of the insert. The left side of the Ek/LIC site is designed to encode the recognition site for enterokinase (DDDDK↓). This feature enables removal of all of the vector-encoded fusion sequences from expressed proteins by cleavage with enterokinase. The following sequences must be added to the 5'-end of the target gene PCR primers to generate vector-compatible overhangs: Sense primer: 5'-GAC GAC GAC AAG ATX*
Antisense primer: 5'-GAG GAG AAG CCC GGT
The antisense primer may encode a stop codon or allow read-through to the vector-encoded stop codon present after the C-terminal tag sequence. * The first nucleotide of the insert-specific sequence must complete the codon ATX to give Met (X = G) or Ile (X = A, C or T). | Time to do procedure • | 50-minute polymerase treatment | • | 5-minute heat inactivation | • | 5-minute annealing | • | 8-minute transformation | • | 60-minute outgrowth |
Features • | Directional cloning into expression vectors | • | Greater than 95% recombinants | • | Complete removal of N-terminal fusion tags with Recombinant Enterokinase |
Ends required • | Any ends (kit creates LIC ends; special primers required) |
Polymerase compatibility |
No Extra Vector-encoded Amino Acids
Amino acids that are not part of the native protein sequence can have an effect on downstream applications, such as activity assays, binding studies, and structural determination. An important feature of the LIC method and the Novagen Ek/LIC vector design is that all aminoterminal vector-encoded sequences can be removed with Recombinant Enterokinase digestion following purification. One Prepared Insert, Many Expression Vector Options
By design, the prepared insert is compatible with every Novagen prepared Ek/LIC vector. 
A Variety of Ek/LIC Vectors Ek/LIC vectors allow protein expression in the following systems:
| | | Bacteria | | • | Powerful T7lac promoter vectors with popular tags for enhanced solubility and purification | | | • | Compatible vectors for coexpression | | | Insect | | • | Vectors for transient, high-level expression without the need for making a recombinant baculovirus | | • | Traditional baculovirus transfer plasmids | | | Mammalian/Multisystem | | • | Vector for high-level expression in bacterial, baculovirus, and mammalian systems |
Coexpression of up to 6 proteins in the same bacterial cell
Two methods are available for coexpression:
| | | pRSF-2 Ek/LIC and pCDF-2 Ek/LIC vectors | | • | contain ColE1-compatible replicons, allowing for coexpression of up to three proteins when both are inserted into the same cell with an ampicillin-resistant pET vector construct | | | LIC Duet™ Adaptors | | • | enable the simultaneous cloning of two open reading frames (ORFs) into any Novagen Ek/LIC vector designed for bacterial protein expression | | | • | allow for coexpression of two proteins from one vector, or up to six proteins with the appropriate vector combinations for transient, high-level expression without the need for making a recombinant baculovirus |
 
Table 1. Vector and host strain compatibilities for coexpression of four to six target proteins
| pET Ek/LIC
(AmpR) | pRSF-2 Ek/LIC
(KanR) | pCDF-2 Ek/LIC
(SmR) | 6 | A | pET
(AmpR) | pRSF-2 Ek/LIC
(KanR) | pCDF-2 Ek/LIC
(SmR) | 5 | A | pET Ek/LIC
(AmpR) | pRSF-2 Ek/LIC
(KanR) | none | 4 | A | pET Ek/LIC
(AmpR) | pCDF-2 Ek/LIC
(SmR) | none | 4 | B | pRSF-2 Ek/LIC
(KanR) | pCDF-2 Ek/LIC
(SmR) | none | 4 | A | | | Group A | (DE3) Subtype | (DE3)pLysS Subtype | | B834(DE3) | B834(DE3)pLysS | | BL21(DE3) | BL21(DE3)pLysS | | BLR(DE3) | BLR(DE3)pLysS | | HMS174(DE3) | HMS174(DE3)pLysS | NovaBlue(DE3)
| | | Rosetta™(DE3) | Rosetta(DE3)pLysS | | Rosetta 2(DE3) | Rosetta 2(DE3)pLysS | | RosettaBlue™(DE3) | RosettaBlue(DE3)pLysS | | Tuner™(DE3) | Tuner(DE3)pLysS | | | | | | | | | | | | |
| Group B | (DE3) Subtype | (DE3)pLysS Subtype | | | B834(DE3) | B834(DE3)pLysS | | | BL21(DE3) | BL21(DE3)pLysS | | | BLR(DE3) | BLR(DE3)pLysS | | | HMS174(DE3) | HMS174(DE3)pLysS | | | NovaBlue(DE3) | | | | Origami™(DE3)‡ | Origami(DE3)pLysS‡ | | | Origami B(DE3) | Origami B(DE3)pLysS | | | Rosetta(DE3) | Rosetta(DE3)pLysS | | | Rosetta 2(DE3) | Rosetta 2(DE3)pLysS | | | RosettaBlue(DE3) | RosettaBlue(DE3)pLysS | | | Rosetta-gami™(DE3)‡ | Rosetta-gami(DE3)pLysS‡ | | | Rosetta-gami B(DE3) | Rosetta-gami B(DE3)pLysS | | Tuner(DE3)
| Tuner(DE3)pLysS | |
| | | | | | | | | | | | † Assumes two target genes are cloned for each Ek/LIC vector using the LIC Duet™ Adaptor method
‡ These strains carry the rpsL mutation that confers resistance to streptomycin; therefore, spectinomycin must be used for selection of pCDFDuet recombinants.
Resistance markers: AmpR, ampicillin/carbenicillin; KanR, kanamycin; SmR, streptomycin/spectinomycin |
E. coli Ek/LIC Vector Kits
Directional PCR cloning into the most powerful E. coli expression vectors
The various pET Ek/LIC Vector configurations are shown below. Configurations of the pCDF-2 and pRSF-2 Ek/LIC Vectors, which are designed for coexpression, are also shown. Ek/LIC vectors for insect and mammalian expression are also available. 
pIEx™ Ek/LIC Vector Kits
Directional PCR cloning into vectors for transient expression in insect cells pIEx™-1, -2 , -3, and -7 are available as Ek/LIC-prepared vectors. These vectors are part of the Radiance™ Cloning and Expression System, which provides expression vectors for efficient directional cloning of PCR products and their subsequent expression in bacterial, insect, or mammalian cells. pBAC™ E. coli Ek/LIC Vector Kits
Efficient, directional cloning of PCR products into pBAC plasmids pBAC™ E. coli LIC Vector Kits are designed for convenient and highly efficient ligation-independent cloning of target genes into pBAC Ek/LIC transfer plasmids in E. coli, starting with inserts amplified with appropriate primers (see The LIC Method and E. coli Ek/LIC Vector Kits earlier in this chapter for more information about LIC.) These vectors are part of the Radiance™ Cloning and Expression System, which provides expression vectors for efficient directional cloning of PCR products and their subsequent expression in bacterial, insect, or mammalian cells. 
- Aslanidis, C. and de Jong, P. J. (1990) Nucleic Acids Res. 18, 6069–6074.
- Haun, R. S., Servanti, I. M., and Moss, J. (1992) BioTechniques 13, 515–518.
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